This invention relates to a human presenilin variant, its encoding cDNA and to the use of these molecules in the diagnosis, prognosis, treatment and evaluation of therapies for cancers and neurodegenerative and immune disorders, particularly early onset Alzheimer""s disease.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Cancer and immune response as a complication of cancer are characterized by continuous cell proliferation, inflammation, and cell death. Several molecular pathways have been linked to these activities, their development and progression. In addition, the analysis of the differential expression of key genes in any of these pathways may be diagnostically or prognostically important. For example, the analysis of cytokine levels is known to be useful as a prognostic indicator for distinguishing between various histologically-similar melanomas (Porter et al. (2001) Ann Surg Oncol 8:116-122).
Alzheimer""s disease (AD) is a degenerative disorder of the central nervous system which causes progressive memory loss and cognitive decline during mid to late adult life and is accompanied by a wide range of neuropathologic features including amyloid deposits and intra-neuronal neurofibrillary tangles. Although the pathogenic pathway leading to neurodegeneration and AD is not well understood, at least three genetic loci that confer genetic susceptibility to the disease have been identified. (Schellenberg, G. D. (1995) Proc. Natl. Acad. Sci. 92:8552-8559; Sherrington, R. et al. (1995) Nature 375:754-760.)
The xcex54 allele (C112 to R) of the apolipoprotein E gene is associated with AD in a significant proportion of late-onset ( greater than 60 years) cases. Mutations in the gene for the xcex2-amyloid precursor protein (xcex2APP) have been found in a small number of families ( less than 3% of cases) with disease onset before 56 years of age. A third locus (AD3) has been mapped by genetic linkage studies to chromosome 14q24.3 and may account for up to 70% of early-onset autosomal-dominant AD. (Sherrington et al. supra.) Although early-onset AD is less common than late-onset AD, the AD3 locus is associated with the most aggressive form of the disease.
Initial studies of known genes on chromosome 14q resulted in their exclusion from the AD3 locus. However, additional studies conducted in a collection of 21 pedigrees segregating AD as a putative autosomal dominant trait resulted in the selection of more than 18 genetic markers associated with the AD3 locus, and the isolation of at least 19 transcripts encoded within this region. (Sherrington et al. supra.) One of these transcripts (S182) was found to encode presenilin-I (PS-1 or 1-467) containing multiple transmembrane domains and resembling an integral membrane protein. A similar gene product (presenilin-II, PS-2) was also identified in association with chromosome 1 in a separate lineage of AD subjects. (Levy-Lahad, E. et al. (1995) Science 269:973-977.) In both PS-1 and PS-2, missense mutations were found that cosegregated with early-onset familial AD in the respective pedigrees. The fact that mutations occurred in conserved domains of the gene and were not found in normal, asymptomatic family members indicates that the mutations are pathogenic for this form of AD. (Sherrington et al. supra; Levy-Lahad et al. supra.) In all cases, the mutations were found in the putative open reading frame of the nucleotide and would be predicted to change the encoded amino acid at that position. Variants of normal human presenilin (PS; 1467, 1463, and 1374) have also been reported. These variants result from either nucleotide deletions or alternative splicing, are ubiquitously expressed, and are associated with intracellular membranes. (Sahara, N. et al. (1996) FEBS Lett. 26:7-11.)
The normal cellular function of PS and, more particularly, the effects of these mutations on cellular function in AD individuals is not yet known. However, the general topology of PS suggests that it is an integral membrane protein such as a receptor, channel protein, or structural membrane protein. In addition, similarities between PS and the Caenorhabditis elegans proteins, SPE-4 and SEL-12, suggest that they may have similar functions. SPE-4 appears to be involved in the transport and storage of soluble and membrane-bound polypeptides during membrane budding and fusion events in C. elegans. (Sherrington et al. supra.) In humans, PS could be involved in similar vesicle transport processes, perhaps in moving xcex2APP. If so, mutations in PS could alter intracellular trafficking of xcex2APP and ultimately lead to altered xcex2APP processing. (Levy-Lahad et al. supra.) Studies using PS-1 and PS-2 and their AD-linked mutations to rescue the effects of a sel-12 mutant in C. elegans demonstrated that mutant human presenilins had reduced ability to rescue the sel-12 mutation relative to PS-1 and PS-2. (Levitan, D et al. (1996) Proc. Natl. Acad. Sci. 93:14940-14944.) The results indicated that the mutant PSs have reduced activity relative to normal PS-1 and PS-2 and that this may be a contributing factor in the development of AD. It was also noted by Levy-Lahad (supra) that several of the amino acid mutations occurring in AD-associated PS were found at or near the beginning of transmembrane domains, and that the mutations may adversely effect the insertion or anchoring of these proteins in the membrane.
The discovery of a human presenilin variant and its encoding cDNA satisfies a need in the art by providing new compositions which are useful in the diagnosis, prognosis, treatment and evaluation of therapies for cancers and neurodegenerative and immune disorders, particularly early onset Alzheimer""s disease.
The present invention is based on the discovery of a human presenilin variant and its encoding cDNA that are differentially expressed in human disorders. The cDNA, protein and an antibody which specifically binds the protein are useful in the diagnosis, prognosis, treatment and evaluation of therapies for cancers and neurodegenerative and immune disorders, particularly early onset Alzheimer""s disease.
The invention provides an isolated cDNA comprising a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ ID NO:1. The invention also provides an isolated cDNA selected from a nucleic acid sequence of SEQ ID NO:2, a fragment of SEQ ID NO:2 selected from SEQ ID NOs:3-7, and a variant selected from SEQ ID NOs:8-11 which has from about 85% to about 91% sequence identity with SEQ ID NO:2, and complements of SEQ ID NOs:2-11. The invention additionally provides compositions, a substrate, and a probe comprising the cDNA or the complement of the cDNA. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make the human presenilin variant. The invention still further provides a transgenic cell line or organism comprising the vector containing a cDNA selected from SEQ ID NO:2-11. The invention additionally provides a fragment, a variant, or the complement of a cDNA selected from SEQ ID NOs:2-11. In one aspect, the invention provides a substrate containing at least one cDNA selected from SEQ ID NOs:2-11 or a complement thereof. In a second aspect, the invention provides a cDNA or the complement thereof which can be used in methods of detection, screening, and purification. In a further aspect, the cDNA is a single-stranded RNA or DNA molecule, a peptide nucleic acid, a branched nucleic acid and the like.
The invention provides a method for using a cDNA to detect differential expression of a nucleic acid in a sample comprising hybridizing a cDNA to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with at least one standard, wherein the comparison indicates differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose transitional cell carcinoma of the bladder and early onset Alzheimer""s disease. In yet another aspect, the cDNA or a fragment or a variant or the complements thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a variant or the complements thereof to screen a library or plurality of molecules or compounds to identify or purify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying or purifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein and a labeling moiety or a pharmaceutical carrier. The invention further provides a method of using the human presenilin variant to treat a subject with cancer or a neurodegenerative or immune disorders comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify or purify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying or purifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with a cancer or a neurodegenerative or immune disorder.
The invention provides a method of using a protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions to form an antibody:protein complex, dissociating the antibody from the protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of a cancer or neurodegenerative or immune disorders.
The invention also provides a method of using a protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which binds specifically to a protein which is expressed in cancers and neurodegenerative and immune disorders, particularly early onset Alzheimer""s disease. The invention also provides a method of using an antibody to diagnose a particular cancer or neurodegenerative disorder comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of the particular cancer or neurodegenerative disorder. The invention further provides a method of using an antibody to treat and immune and neurodegenerative disorders comprising administering to a patient in need of such treatment a composition comprising the purified antibody and a pharmaceutical carrier.
The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA of SEQ ID NO:8-1 1, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.